Title: Purple Proteins: Expressing, Purifying, and Characterizing the pGEM-gbr22 Protein
Make intro more relevant to this lab Introduction:
Successfully overexpressing a protein is useful in biological and biomedical research. There are many uses in expressing recombinant proteins that allow researchers to analyze and perhaps manipulate these proteins to work towards a specific function. For example, a research group led by Bayer-Giraldi worked to overexpress an antifreeze protein under different climate conditions in order to study the efficiency of the protein’s ability to inhibit ice recrystallization [1]. When it comes to expressing recombinant proteins there are a variety of approaches unique to the type of protein utilized. In a review published by Nature Methods, a group of researchers pooled together their personal experience and wrote a “beginner’s guide” to recombinant proteins, discussing topics such as the type of host to utilize, purification strategies, and cloning sites [2]. The objective of this lab was to overexpress, purify, and characterize the recombinant purple protein pGEM-gbr22 tagged with hexahistidine tag and then inserting the DNA plasmid into a recombinant host Eschericihia coli (BL21-DE3). The cells were cultured overnight and purified through a Ni-NTA column chromatography that would bind to the histidine tag. To affirm that the pGEM-gbr22 protein was only collected, the protein was characterized by SDS-PAGE gel electrophoresis that works by running a charge through an acrylamide gel to separate proteins present based on size. After successful expression, purification, and characterization, the pGEM-gbr22 protein should produce a band at 25.79 kDa, according to the ExPASy Bioinformatics Resource Portal (needed to cite this).
Materials & Methods:
Protein Expression:
In order to overexpress the protein pGEM-gbr22 in Escherichia coli, twenty-five (Just write 25) µL of competent bacterial cells (BL21-DE3) (need to cite the company & state) and 2 µL of DNA plasmid were combined and placed on ice for 30 minutes and heat shocked for 45 seconds in a 42°C water bath. 200 µL SOC media was then added to the cell mixture. After 30 minutes in the 37°C shaking incubator, 50 µL of bacteria mixture were dispensed on Ampicillin-positive agar plate and stored in the 37°C incubator overnight. A control plate was generated using the same technique but excluded the DNA plasmid.
After overnight incubation, one bacterial colony was transferred into LB broth/Ampicillin media and placed in the 37°Celsius shaking incubator for 8 hours. After incubation, 0.625 mL of the bacterial culture was transferred into an Erlenmeyer flask with LB broth/ampillicin media, and placed back into the shaking incubator for 21 hours.
The next day, 500 µL of the cell culture was centrifuged in the Allegra X-15 Benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) for 10 minutes at 5000 rpm at 4°C. The supernatant of the cell mixture was disposed in bleach and the cell pellet was weighed. The cell pellet was then resuspended in 2.5 mL 1x PBS with 50μL 50 μg/μL lysozyme for a final concentration (1mg/mL). The cell mixture was placed in the -20°C freezer for storage.
Protein Purification:
To purify the cells, 2 µL cyanase (added benzonase) was added to the defrosted cell mixture and then was dispensed into two 1.7 Eppendorf tubes to be centrifuged for 20 minutes at 14,000 rpm. 50 µL of the supernatant was saved, labeled “Sample 2,” and the rest of the supernatant was disposed. The lysate was filtered through a 0.45 µm syringe and then 0.5 mL Ni-NTA resin/buffer mix was added. The cell mixture was then transferred into a Bio-Rad chromatography Econo column and a 50 µL of the flowthrough sample was taken. Then 5 mL 20mM Imidazole/1X PBS solution was dispensed into the column and another 50 µL sample was taken. Then 5 mL 250mM Imidazole/1X PBS solution was dispensed into the column and another sample was taken. The elution step was repeated once more. The Nanodrop spectrophotometer (need to state company & state) was used to measure the concentration and yield of the eluted samples at 280 nm.
Protein Characterization:
The six samples collected during the expression and purification steps were analyzed through gel electrophoresis. Sample 1 (cell mixture) was prepared by adding 500 µL Nanopure water and centrifugation for 5 minutes at 5000 rpm. The spent media was removed and disposed, and the cell pellet was resuspended in 200 µL Nanopure and 40 µL loading buffer. Then 10μL 6x loading buffer was added to Sample 2-6 and all samples were heat shocked for 5 minutes at 95°C. The pre-cast gel (4-20%) was set up in the mini-PROTEAN tank with 500mL 1X TGS and the wells were loaded (in order) 7 µL molecular weight standard, 20 µL of the six samples in separate wells, and another 7 µL molecular weight standard. The gel was run for 25 minutes at 200V. The gel was then rinsed in 100 mL Nanopure three times, stained with Imperial Protein Stain for 1.5 hours, and rinsed twice again with Nanopure. Then the gel was left to wash in the orbital shaker in 100 mL overnight. After this rinse, the gel was covered in Waltman paper, dried and then analyzed.
Results:
Fig 1: Ampicillin positive agar plate with BL21(DE3) and without pGEM-gbr22 (DNA control) after 16 hours in 37 degree Celsius shaking incubator, showing no apparent bacterial growth
Fig 2: Ampicillin positive agar plate with BL21(DE3) and pGEM-gbr22 DNA plasmid after 16 hours in 37 degree Celsius shaking incubator
Fig 3: "Fun" plate that was coughed on but showed no apparent bacterial growth after 16 hours in 37 degree Celsius shaking incubator
Two colonies were collected from the agar plates overall, but only one cell mixture was used for purification.
Fig 4: LB broth and ampicillin bacteria mixture of bacteria BL21(DE3) and DNA pGEM-gbr22 after 20 hours in incubation at 37 degrees Celsius
Fig 5: Wet cell pellet of bacteria BL21(DE3) with successful expression of DNA pGEM-gbr22 after 20 hours in incubation (37 degree Celsius). Total mass of 0.37 grams obtained.
After overexpressing the pGEM-gbr22 protein in Escherichia coli (BL21-DE3), 0.37 grams of protein-expressing E. coli were collected and 0.757 mg protein collected overall.
Fig. 6: First elution of approximately 5 mL of pGEM-gbr22 containing 1x PBS buffer and 250mM Imidazole solution after Ni-NTA affinity purification
Fig. 7: Second elution of approximately 5mL of pGEM-gbr22 containing 1x PBS buffer and 250mM Imidazole solution after Ni-NTA affinity purification
The yield of protein after purification was 0.757 mg pGEM-gbr22.
Fig 8: Protein (pGEM-gbr22) absorbance at 280 nm for Elution 1, Trial 1
Concentration of Elution 1 using Beer’s Law and Nanodrop Spectroscopy data
A= Ebc
0.228 = (38850 M-1 cm-1)(1 cm) (c)
C = 5.302 x 10-6 mol/L * 1L/1000mL * 25794.2 g/mol * 1000 mg/1g = 0.1514 mg/mL
Fig 9: SDS-PAGE band profile of Thermo Scientific PageRuler Prestained Protein Ladder measuring from 10-170 kDa.
Fig 10: SDS-PAGE Gel characterizing gbr22 protein. (From left to right: Molecular Weight Standard; Cell Sample; Soluble fraction; Syringe filtered flowthrough; Wash Flowthrough; Elution 1 Flowthrough; Elution 2 Flowthrough)
Caption should look like this: Lane 1: Lane 2: Lane 3: This is easier to read
Discussion:
In order to properly express the gene and assure proper technique was utilized, an ampicillin resistant gene was included into the sequence of the gbr22 protein used so that if there was a lack of growth of E. coli colonies on the agar plates, it would signal improper intake of the DNA plasmid. The antibiotic ampicillin would easily kill E. coli bacteria not expressing the purple protein. After successful expression, the first step in purifying the protein, lysozyme was added to lyse/break down the cell wall of the bacteria and release the protein into solution. To decrease the viscosity of the mixture, cyanase (benzonase) was added to digest the RNA/DNA present.
An important aspect of purifying the gbr22 protein through Ni/NTA column chromatography was that the protein was tagged with a hexa-histidine tag (where did the hexa-histidine tag exist on the protein sequences? N or C terminus?) that would bind to the large beads coated in nickel ion. Because imidazole is similar to histidine in structure, it competes to bind itself to the nickel ion. The wash buffer had a low concentration of imidazole and worked to rid of loosely bound molecules in the column. The elution buffer had a much higher concentration of imidazole and when added, would elute the protein off of the column into the flowthrough.
When analyzing the samples collected during protein purification, each individual sample was a sample taken after each step of purification. Sample 1 was the original unpurified cell mixture. Sample 2 contained the soluble fraction of the cell mixture after centrifugation. Sample 3 contained the flowthrough of the cell solution after syringe filtration. Sample 4 was the flowthrough of the Ni-NTA chromatography after wash buffer was added. Sample 5 and Sample 6 contained the flowthrough sample after consecutive rounds of the elution buffer.
In regards to the size and purity of the protein, these questions cannot be answer due to a possible loading error of the gel. After the gel was run, stained, and cleaned, there were no bands present in the column marked for Elution 1 where most of the gbr22 protein should be present. It may be possible that while loading the Elution 1 sample into the gel, it fell into solution because there was not enough glycerol. It was concluded that this error did not occur due to running the same sample in two different wells because then that would produce two identical columns rather than the lack thereof.
It is important to note though that although the protein was nonexistent in Elution 1, there were two faint bands present in Elution 2. If Elution 1 was present, the same two bands could have also been present in Elution 1 but more concentrated. This means that the gbr22 protein in solution was impure due to the presence of an unknown protein.
Other possible of sources of error in expressing, purifying, and characterizing the protein may be improperly made solutions or overall sterile techniques. The various solutions used in this lab (PBS buffer, Imidazole, TGS buffer, etc) may have been made incorrectly so that the concentration needed for this lab was not the concentration actually made. For example, improperly made Imidazole that was at a lower concentration than needed would not be as effective at eluting the gbr22 protein off of the Ni-NTA column. This error may be relevant to the appearance of a foreign protein in solution because the imidazole was not concentrated enough to elute proteins that were more tightly bound to the nickel ion resin in the wash step.
Conclusions:
After expressing the pGEM-gbr protein in the host Escherichia coli (BL21-DE3), purifying the protein from the cell, and then characterizing the protein, 0.757 mg of pGEM-gbr22 protein were collected although the purity of this sample is unknown. In the future of Virtual Drug Screening, knowing and understanding these techniques will be vital in target research by manipulating the recombinant protein to accomplish a certain function and then inserting the protein in a recombinant host to determine its effect. Future direction: Enzyme & Inhibition Assay References:
[1] Bayer-Giraldi, M.; Weikusat, I.; Besir, H.; Dieckmann, G., Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice. Cryobiology2011,63 (3), 210-9. [2] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods2008,5 (2), 135-46.
Purple Proteins: Expressing, Purifying, and Characterizing the pGEM-gbr22 Protein
Make intro more relevant to this lab
Introduction:
Successfully overexpressing a protein is useful in biological and biomedical research. There are many uses in expressing recombinant proteins that allow researchers to analyze and perhaps manipulate these proteins to work towards a specific function. For example, a research group led by Bayer-Giraldi worked to overexpress an antifreeze protein under different climate conditions in order to study the efficiency of the protein’s ability to inhibit ice recrystallization [1]. When it comes to expressing recombinant proteins there are a variety of approaches unique to the type of protein utilized. In a review published by Nature Methods, a group of researchers pooled together their personal experience and wrote a “beginner’s guide” to recombinant proteins, discussing topics such as the type of host to utilize, purification strategies, and cloning sites [2]. The objective of this lab was to overexpress, purify, and characterize the recombinant purple protein pGEM-gbr22 tagged with hexahistidine tag and then inserting the DNA plasmid into a recombinant host Eschericihia coli (BL21-DE3). The cells were cultured overnight and purified through a Ni-NTA column chromatography that would bind to the histidine tag. To affirm that the pGEM-gbr22 protein was only collected, the protein was characterized by SDS-PAGE gel electrophoresis that works by running a charge through an acrylamide gel to separate proteins present based on size. After successful expression, purification, and characterization, the pGEM-gbr22 protein should produce a band at 25.79 kDa, according to the ExPASy Bioinformatics Resource Portal (needed to cite this).
Materials & Methods:
Protein Expression:
In order to overexpress the protein pGEM-gbr22 in Escherichia coli, twenty-five (Just write 25) µL of competent bacterial cells (BL21-DE3) (need to cite the company & state) and 2 µL of DNA plasmid were combined and placed on ice for 30 minutes and heat shocked for 45 seconds in a 42°C water bath. 200 µL SOC media was then added to the cell mixture. After 30 minutes in the 37°C shaking incubator, 50 µL of bacteria mixture were dispensed on Ampicillin-positive agar plate and stored in the 37°C incubator overnight. A control plate was generated using the same technique but excluded the DNA plasmid.
After overnight incubation, one bacterial colony was transferred into LB broth/Ampicillin media and placed in the 37°Celsius shaking incubator for 8 hours. After incubation, 0.625 mL of the bacterial culture was transferred into an Erlenmeyer flask with LB broth/ampillicin media, and placed back into the shaking incubator for 21 hours.
The next day, 500 µL of the cell culture was centrifuged in the Allegra X-15 Benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) for 10 minutes at 5000 rpm at 4°C. The supernatant of the cell mixture was disposed in bleach and the cell pellet was weighed. The cell pellet was then resuspended in 2.5 mL 1x PBS with 50μL 50 μg/μL lysozyme for a final concentration (1mg/mL). The cell mixture was placed in the -20°C freezer for storage.
Protein Purification:
To purify the cells, 2 µL cyanase (added benzonase) was added to the defrosted cell mixture and then was dispensed into two 1.7 Eppendorf tubes to be centrifuged for 20 minutes at 14,000 rpm. 50 µL of the supernatant was saved, labeled “Sample 2,” and the rest of the supernatant was disposed. The lysate was filtered through a 0.45 µm syringe and then 0.5 mL Ni-NTA resin/buffer mix was added. The cell mixture was then transferred into a Bio-Rad chromatography Econo column and a 50 µL of the flowthrough sample was taken. Then 5 mL 20mM Imidazole/1X PBS solution was dispensed into the column and another 50 µL sample was taken. Then 5 mL 250mM Imidazole/1X PBS solution was dispensed into the column and another sample was taken. The elution step was repeated once more. The Nanodrop spectrophotometer (need to state company & state) was used to measure the concentration and yield of the eluted samples at 280 nm.
Protein Characterization:
The six samples collected during the expression and purification steps were analyzed through gel electrophoresis. Sample 1 (cell mixture) was prepared by adding 500 µL Nanopure water and centrifugation for 5 minutes at 5000 rpm. The spent media was removed and disposed, and the cell pellet was resuspended in 200 µL Nanopure and 40 µL loading buffer. Then 10μL 6x loading buffer was added to Sample 2-6 and all samples were heat shocked for 5 minutes at 95°C. The pre-cast gel (4-20%) was set up in the mini-PROTEAN tank with 500mL 1X TGS and the wells were loaded (in order) 7 µL molecular weight standard, 20 µL of the six samples in separate wells, and another 7 µL molecular weight standard. The gel was run for 25 minutes at 200V. The gel was then rinsed in 100 mL Nanopure three times, stained with Imperial Protein Stain for 1.5 hours, and rinsed twice again with Nanopure. Then the gel was left to wash in the orbital shaker in 100 mL overnight. After this rinse, the gel was covered in Waltman paper, dried and then analyzed.
Results:
Two colonies were collected from the agar plates overall, but only one cell mixture was used for purification.
After overexpressing the pGEM-gbr22 protein in Escherichia coli (BL21-DE3), 0.37 grams of protein-expressing E. coli were collected and 0.757 mg protein collected overall.
The yield of protein after purification was 0.757 mg pGEM-gbr22.
Concentration of Elution 1 using Beer’s Law and Nanodrop Spectroscopy data
A= Ebc
0.228 = (38850 M-1 cm-1)(1 cm) (c)
C = 5.302 x 10-6 mol/L * 1L/1000mL * 25794.2 g/mol * 1000 mg/1g = 0.1514 mg/mL
Caption should look like this:
Lane 1:
Lane 2:
Lane 3:
This is easier to read
Discussion:
In order to properly express the gene and assure proper technique was utilized, an ampicillin resistant gene was included into the sequence of the gbr22 protein used so that if there was a lack of growth of E. coli colonies on the agar plates, it would signal improper intake of the DNA plasmid. The antibiotic ampicillin would easily kill E. coli bacteria not expressing the purple protein. After successful expression, the first step in purifying the protein, lysozyme was added to lyse/break down the cell wall of the bacteria and release the protein into solution. To decrease the viscosity of the mixture, cyanase (benzonase) was added to digest the RNA/DNA present.
An important aspect of purifying the gbr22 protein through Ni/NTA column chromatography was that the protein was tagged with a hexa-histidine tag (where did the hexa-histidine tag exist on the protein sequences? N or C terminus?) that would bind to the large beads coated in nickel ion. Because imidazole is similar to histidine in structure, it competes to bind itself to the nickel ion. The wash buffer had a low concentration of imidazole and worked to rid of loosely bound molecules in the column. The elution buffer had a much higher concentration of imidazole and when added, would elute the protein off of the column into the flowthrough.
When analyzing the samples collected during protein purification, each individual sample was a sample taken after each step of purification. Sample 1 was the original unpurified cell mixture. Sample 2 contained the soluble fraction of the cell mixture after centrifugation. Sample 3 contained the flowthrough of the cell solution after syringe filtration. Sample 4 was the flowthrough of the Ni-NTA chromatography after wash buffer was added. Sample 5 and Sample 6 contained the flowthrough sample after consecutive rounds of the elution buffer.
In regards to the size and purity of the protein, these questions cannot be answer due to a possible loading error of the gel. After the gel was run, stained, and cleaned, there were no bands present in the column marked for Elution 1 where most of the gbr22 protein should be present. It may be possible that while loading the Elution 1 sample into the gel, it fell into solution because there was not enough glycerol. It was concluded that this error did not occur due to running the same sample in two different wells because then that would produce two identical columns rather than the lack thereof.
It is important to note though that although the protein was nonexistent in Elution 1, there were two faint bands present in Elution 2. If Elution 1 was present, the same two bands could have also been present in Elution 1 but more concentrated. This means that the gbr22 protein in solution was impure due to the presence of an unknown protein.
Other possible of sources of error in expressing, purifying, and characterizing the protein may be improperly made solutions or overall sterile techniques. The various solutions used in this lab (PBS buffer, Imidazole, TGS buffer, etc) may have been made incorrectly so that the concentration needed for this lab was not the concentration actually made. For example, improperly made Imidazole that was at a lower concentration than needed would not be as effective at eluting the gbr22 protein off of the Ni-NTA column. This error may be relevant to the appearance of a foreign protein in solution because the imidazole was not concentrated enough to elute proteins that were more tightly bound to the nickel ion resin in the wash step.
Conclusions:
After expressing the pGEM-gbr protein in the host Escherichia coli (BL21-DE3), purifying the protein from the cell, and then characterizing the protein, 0.757 mg of pGEM-gbr22 protein were collected although the purity of this sample is unknown. In the future of Virtual Drug Screening, knowing and understanding these techniques will be vital in target research by manipulating the recombinant protein to accomplish a certain function and then inserting the protein in a recombinant host to determine its effect.
Future direction: Enzyme & Inhibition Assay
References:
[1] Bayer-Giraldi, M.; Weikusat, I.; Besir, H.; Dieckmann, G., Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice. Cryobiology 2011, 63 (3), 210-9.
[2] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.